1. Field of the Invention
The present invention relates to compositions and methods of therapeutic use of PEGylated cytokines, such as interferon-alpha (IFN-α), more preferably IFN-α2b. However, the skilled artisan will realize that the invention is not so limited and more broadly covers PEGylated forms of other immunomodulators or different therapeutic agents. Preferably, the PEGylated compositions are made using the dock-and-lock (DNL) technique, as exemplified in U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference. The PEGylated cytokines, immunomodulators and other therapeutic agents retain in vitro activity and preferably show enhanced in vivo efficacy and increased serum half-life. Additional advantages of the PEGylated products may also include lower immunogenicity, decreased dosing frequency, increased solubility, enhanced stability, and reduced renal clearance.
2. Related Art
Interferon
Interferon-α (IFNα) has been reported to have anti-tumor activity in both animal models of cancer (Ferrantini et al., 1994, J Immunol 153:4604-15) and human cancer patients (Gutterman et al., 1980, Ann Intern Med 93:399-406). IFNα can exert a variety of direct anti-tumor effects, including down-regulation of oncogenes, up-regulation of tumor suppressors, enhancement of immune recognition via increased expression of tumor surface MHC class I proteins, potentiation of apoptosis, and sensitization to chemotherapeutic agents (Gutterman et al., 1994, PNAS USA 91:1198-205; Matarrese et al., 2002, Am J Pathol 160:1507-20; Mecchia et al., 2000, Gene Ther 7:167-79; Sabaawy et al., 1999, Int J Oncol 14:1143-51; Takaoka et al, 2003, Nature 424:516-23). For some tumors, IFNα can have a direct and potent anti-proliferative effect through activation of STAT1 (Grimley et al., 1998 Blood 91:3017-27).
Indirectly, IFNα can inhibit angiogenesis (Sidky and Borden, 1987, Cancer Res 47:5155-61) and stimulate host immune cells, which may be vital to the overall antitumor response but has been largely under-appreciated (Belardelli et al., 1996, Immunol Today 17:369-72). IFNα has a pleiotropic influence on immune responses through effects on myeloid cells (Raefsky et al, 1985, J Immunol 135:2507-12; Luft et al, 1998, J Immunol 161:1947-53), T-cells (Carrero et al, 2006, J Exp Med 203:933-40; Pilling et al., 1999, Eur J Immunol 29:1041-50), and B-cells (Le et al, 2001, Immunity 14:461-70). As an important modulator of the innate immune system, IFNα induces the rapid differentiation and activation of dendritic cells (Belardelli et al, 2004, Cancer Res 64:6827-30; Paquette et al., 1998, J Leukoc Biol 64:358-67; Santini et al., 2000, J Exp med 191:1777-88) and enhances the cytotoxicity, migration, cytokine production and antibody-dependent cellular cytotoxicity (ADCC) of NK cells (Biron et al., 1999, Annu Rev Immunol 17:189-220; Brunda et al. 1984, Cancer Res 44:597-601).
The therapeutic effectiveness of IFNs has been validated to date by the approval of IFN-α2 for treating hairy cell leukemia, chronic myelogenous leukemia, malignant melanoma, follicular lymphoma, condylomata acuminata, AIDs-related Kaposi sarcoma, and chronic hepatitis B and C; IFN-β for treating multiple sclerosis; and IFN-γ for treating chronic granulomatous disease and malignant osteopetrosis. Despite a vast literature on this group of autocrine and paracrine cytokines, their functions in health and disease are still being elucidated, including more effective and novel forms being introduced clinically (Pestka, 2007, J. Biol. Chem. 282:20047-51; Vilcek, 2006, Immunity 25:343-48).
Therapy of viral infection with IFNs has been reported for a wide variety of viral species. The combination of IFN-α and IFN-γ or IFN-β and IFN-γ was reported to inhibit replication of dengue virus (Diamond & Harris, 2001, Virology 289:297-311). Low dose prophylactic oral administration of IFN-α protected against a lethal challenge with influenza virus (Belharz et al., 2007, Biochem Biophys Res Commun 355:740-44). Similarly, prophylactic IFN-α-2 reduced rhinovirus-induced infection (Morey & Blackwell, 1989, Aviat Space Environ Med 60:1028). The combination of IFN-α-6 and IFN-β was reported to synergistically reduce infection with cytomegalovirus when administered prophylactically. A synergistic effect of IFN-β and IFN-γ was also reported for reducing acyclovir-resistant herpes simplex viral infection (Huang et al., 2010, J Gen Virol 91:591-98). IFN-γ was observed to inhibit DNA synthesis of vaccinia virus in macrophages (Melkova & Esteban, 1994, Virology 198:731-35). An IFN-γ mimetic peptide prevented encephalomyocarditis virus infection in both tissue culture and in vivo in mice (Mujtaba et al., 2006, Clin Vaccine Immunol 13:944-52).
The promise of IFNα as a cancer therapeutic has been hindered primarily due to its short circulating half-life and systemic toxicity. As demonstrated by PEGINTRON® (Grace et al., 2001, J. Interferon Cytokine Res 21:1103-15) and PEGASYS® (Bailon et al., 2001, Bioconjugate Chem 12:195-202), the efficacy of IFNs can be enhanced by improving their bioavailability with PEGylation, with the resulting conjugate exhibiting an increased serum half-life (Harris and Chess, 2003, Nat Rev Drug Discov 2:214-21). Additional advantages of PEGylation in general include reduced immunogenicity, decreased dosing frequency, increased solubility, enhanced resistance to proteolysis, and exclusion of renal clearance.
PEGylation
Because the most common reactive sites on proteins (including peptides) for attaching PEG are the c amino groups of lysine and the a amino group of the N-terminal residue, early methods of PEGylation resulted in modification of multiple sites, yielding not only monoPEGylated conjugates consisting of mixtures of positional isomers, such as PEGINTRON®™ (Grace et al., J. Biol. Chem. 2005; 280:6327) and PEGASYS®® (Dhalluin et al., Bioconjugate Chem. 2005; 16:504), but also adducts comprising more than one PEG chain. Site-specific attachment of a single PEG to the α amino group of the N-terminal residue was reported to be the predominant product upon reacting PEG-aldehyde (PEG-ALD) at low pH with IFN-β1b (Basu et al., Bioconjugate Chem. 2006; 17:618) or IFN-β1a (Pepinsky et al., J. Pharmacol. Exp. Ther. 2001, 297:1059). Similar strategies were applied to prepare N-terminally linked PEG to G-CSF (Kinstler et al., Pharm. Res. 1996; 13:996) or type I soluble tumor necrosis factor receptor (Kerwin et al., Protein Sci. 2002; 11:1825). More recently, a solid-phase process for PEGylation of the N-terminus of recombinant interferon alpha-2a was reported (Lee et al., Bioconjug. Chem. Oct. 18, 2007, epub).
Site-directed PEGylation of a free cysteine residue introduced into a target protein has also been achieved with PEG-maleimide (PEG-MAL) for several recombinant constructs including IL-2 (Goodson and Katre, Biotechnology. 1990:8:343); IFN-α2 (Rosendahl et al., Bioconjugate Chem. 2005; 16:200); GM-CSF (Doherty et al., Bioconjugate Chem. 2005; 16:1291); scFv (Yang et al., Protein Eng. 2003; 16:761), and miniantibodies (Kubetzko et al., J. Biol. Chem; 2006; 201:35186). A popular approach for improving the therapeutic efficacy of an enzyme has been to prepare conjugates containing multiple PEG of small size, as known for methioninase (Yang et al., Cancer Res. 2004; 64:6673); L-methionine γ-lyase (Takakura et al., Cancer Res. 2006:66:2807): arginine deaminase (Wang et al., Bioconjugate Chem. 2006; 17:1447); adenosine deaminase (Davis et al., Clin. Exp. Immunol. 1981, 46:649); L-asparaginase (Bendich et al., Clin. Exp. Immunol. 1982, 48:273); and liver catalase (Abuchowski et al., J. Biol. Chem. 1977, 252:3582).
PEGylations of bovine serum albumin (Abuchowski et al., J. Biol. Chem. 1977; 252:3578); hemoglobin (Manjula et al., Bioconjugate Chem. 2003; 14:464); visomant (Mosharraf et al., Int. J. Pharm. 2007; 336:215); small molecules such as inhibitors of integrin α4β1 (Pepinsky et al., J. Pharmacol. Exp. Ther. 2005, 312:742); lymphoma-targeting peptides (DeNardo et al., Clin. Cancer. Res. 2003; 9(Suppl.):3854s); anti-VEGF aptamer (Bunka and Stockley, Nat. Rev. Microbiol. 2006; 4:588) and oligodeoxynucleotides (Fisher et al., Drug Metab. Dispos. 2004; 32:983) have also been described. The feasibility of reversible or releasable PEGylation, wherein covalently attached PEG can be cleaved in vivo, has been shown with a variety of degradable linkages, exemplified by linking PEG-SH to IFN-α2 with a 2-sulfo-9-fluorenylmethoxycarbonyl-containing bifunctional reagent (Peleg-Shulman et al., 2004, J. Med Chem 47:4897-4904), by attaching linear or branched PEG-BCN3 to lysozyme or IFN-β1b (Zhao et al., 2006, Bioconjugate Chem 17:341-51), or by conjugating PEG to lysozyme via a dithiobenzyl urethane linkage (Zalipsky et al., 2007, Bioconjugate Chem 18:1869-78). Recently, a strategy for site-specific PEGylation of disulfide bonds has been reported (Shaunak et al., 2006, Nat Chem Biol 2:312-13), but its use may be limited to only those proteins with native disulfide bonds that are suitably oriented for such modification.
There exists a need for a general method of PEGylation that would produce a monoPEGylated or a biPEGylated conjugate linked site-specifically to a predetermined location of a therapeutic agent such as a cytokine, which retains the bioactivity of the unmodified agent. A further need exists for PEG-cytokine conjugates that exhibit improved in vivo efficacy, decreased toxicity and/or superior pharmacokinetic properties.